Species of Dirofilaria (Spirurida, Onchocercidae) are vector-borne filarioid nematode parasites. Worldwide, filarioid nematodoses with major public health importance include lymphatic filariosis and onchocercosis, while dirofilariosis, setariosis and onchocercosis cause major animal health hazards and have economic implications [1]. Animal parasitic filarioids are often also zoonotic. There is recent evidence documenting the expansion of some animal diseases associated with filarioid parasites to boreal and sub-Arctic areas, including Finland [2, 3], and at northern latitudes, species of several filarioid genera are now known to be emerging [4, 5].
Dirofilaria immitis, the dog heartworm, and Dirofilaria repens, the canine subcutaneous worm, use mosquitoes as vectors. The host specificity of these parasites is not high and they are found occasionally in foxes and cats as well as sporadically in other mammal species, including humans [6, 7]. Since potential mosquito vectors are available virtually over all the sub-Arctic, and many arctic regions, the northern border of distribution of these Dirofilaria species is not determined by the availability of the vectors but rather by the ability of microfilariae to mature into infectious larvae in the mosquito vector [8,9,10], as maturation is temperature-dependent. Dirofilaria repens development in mosquitoes to the infective stage takes 8–13 days at 28–30 °C, 10–11 days at 26 °C and 16–20 days at 22 °C [11]. At temperatures below 14 °C, D. immitis larval development ceases [12], but restarts if the temperature rises. When a mosquito with infective stage (L3) larvae feeds on a human being, it may infect this aberrant host [13]. Both subcutaneous, periorbital and eyelid human infections caused by D. repens occur in endemic areas [14].
Dirofilaria repens is endemic in dogs in Africa, southeastern Asia, and the Mediterranean region [15]. In Europe, its distribution range has recently expanded northward, assumedly partly due to climate change [8, 16,17,18]. In a 2012 publication, the European Scientific Counsel Companion Animal Parasites [19] reported the parasite to have established in Austria, Germany and Poland. It has also been found in Lithuania in dogs [20], and in Latvia, in both dogs and humans [21, 22]. Thus far, the northernmost European site where the parasite life-cycle has been confirmed to take place is Estonia, where D. repens microfilaraemia was seen in three dogs in 2013–2014 [23]. In European Russia, the reported northern limit of D. repens is in the Novgorod Region [24]. According to Russian veterinary practitioners, however, dirofilariosis occurs in the dog population of Saint Petersburg, as was also reported on a veterinary clinic website (Anna Nikanorova, pers. comm. 2017). The Novgorod Region and the southern part of the Leningrad Region are located approximately at the same latitude as Estonia (Fig. 1). Recently, three cases of human ocular dirofilariosis were reported from Saint Petersburg [25, 26]. In one case, the patient had reportedly not left the region during the previous three years, suggesting endemicity of D. repens in the Leningrad Region. In addition, a single case of dirofilariosis was reported in 1999 in the city of Arkhangelsk [27]. A 15 year-old child was suspected to have been infected in Arkhangelsk, but because of travel history to Sochi and Rostov-on-Don, the origin of infection could not be confirmed. In Yakutsk, Siberia, located over permafrost and known as the coldest city on earth, three D. repens infections were reported in dogs and one in a cat [28]. Moreover, six cases of D. immitis infection were diagnosed in dogs and one in a fox between 1996 and 2010 [28].
In Finland, autochthonous D. repens cases in dogs have not been diagnosed previously, but a dog imported from Romania was found infected in 2014 [29]. Another species, Dirofilaria ursi, a parasite of the brown bear, using black flies as vectors and having lower temperature requirements for development in its vector [30], is prevalent in Finnish bears at least in Northern Karelia (unpublished pers. obs.).
Human dirofilariosis has been increasingly reported in the past few years and thus should be considered an emerging zoonosis in many parts of the world [13, 31, 32]. Here, we report in a human being the first autochthonous D. repens infection in Finland. The infected patient lived in Hamina, southeastern Finland (Fig. 1), which is around 35 km from the Russian border. We document the surgical removal of the parasite nodule and the identification of the adult, gravid female nematode and its microfilariae. Further, we discuss possible transmission pathways. Moreover, we report two cases in dogs imported to Finland from St Petersburg and Vyborg, Russia, the latter just 25 km from the Finnish border and 90 km from Hamina.
Autochthonous human case, southeastern Finland
In March 2015, a 70-year-old Finnish woman, with no recent travel history abroad, sought medical care due to erythema, swelling and some itching on the volar side of her left forearm. Suspecting an allergic reaction, the general practitioner prescribed a single dose of oral corticosteroid and a course of antihistamine. After four days, antibacterial cephalexin chemotherapy was initiated because the symptoms had not abated and painful nodules were observed in the forearm. The absolute leucocyte count (13.1 × 109/l; norm 3.4–8.2 × 109/l), eosinophil leukocyte count (1.2 × 109/l; norm 0.03–0.1 × 109/l) and C-reactive protein concentration (22 mg/l; norm <3 mg/l) were elevated. Soon after starting cephalexin, the patient got a possible allergic reaction, with flush over the face and upper chest, and the antibiotic was replaced with piperacillin and tazobactam for three days and, after that, continued with clindamycin. Over the next two weeks, the pain and swelling disappeared, but a thumb tip size nodule remained in the forearm. In early April, 20 days after the first visit to the general practitioner, the nodule was surgically removed for histological examination. The excision wound healed normally leaving only a minor scar.
Upon histological study, an inflammatory reaction composed of eosinophilic granulocytes and histiocytes surrounding a nematode was noted. Morphology revealed at least one gravid, adult female filarioid nematode, having a cuticle with evenly spaced external longitudinal cuticular ridges. The musculature was coelomyarian (muscle fibers in a cylindrical pattern) and polymyarian (many cells in each quadrant of a cross-section), and the intestine was very small (Fig. 2). The nematode had two large uteri filled with microfilariae. These findings were characteristic of Dirofilaria spp. The external longitudinal cuticular ridges ruled out D. immitis, but we could not distinguish between other potential species. Since D. repens is present in Estonia, and the brown bear parasite D. ursi is prevalent in eastern Finland, we aimed at specific diagnosis by both morphological examination of microfilariae and molecular identification.
Three weeks after the parasitic nodule had been excised, diurnal and nocturnal whole heparine blood samples were collected for microscopic analysis of possible microfilariae, concentrated using a standard filtration method with Millipore filters [33], and stained on microscope slides with Giemsa. In the nocturnal samples, microfilariae were detected with a maximum of 2 mf/ml. Two months after the excision, 1 mf/8 ml was still detected, and in August, 4.5 months after the excision, no microfilariae were found from the patient.
The microfilariae possessed 2 or 3 nuclei in the cephalic space (Fig. 2). The length of 10 microfilariae was measured, which was not easy since caudal filaments of crooked microfilariae were not clearly visible (Fig. 2). Length measurements, 200–260 μm, agreed with that reported for D. ursi in bears (198–242 μm) [34]. Microfilariae of D. repens in dogs have been reported to be longer (323 ± 21 μm) [35], or, using a somewhat different Knott’s method, even longer (369 ± 11 μm) [36].
Infection in two dogs imported from Vyborg and St Petersburg
We also report here two confirmed cases in dogs imported to Finland from Russia, one from Vyborg and another from St Petersburg, located about 25 km and 160 km from the Finnish border, respectively.
In May 2013, a 2 year-old Rabbit Dachshund, imported from St Petersburg, Russia, more than a year earlier, was brought to a veterinary clinic in Turku with vomiting and presenting signs of abdominal pain. Laboratory examinations revealed, apparently unrelated to the presented illness, many mf in a Giemsa-stained thin blood film. Microfilariae were about 370 μm in length, possessed rather short cephalic space terminating by a pair of nuclei separate from the remaining somatic nuclei of the microfilaria. These morphological features were considered typical of D. repens.
In 2015, a 15 month-old mixed breed bitch imported from Vyborg was presented for a routine ovariohysterectomy in Helsinki. During the surgery, the veterinary surgeon found a wormlike organism about 2 cm long squirming in the abdominal cavity. The surgeon preserved the worm in alcohol and sent it for identification. Based on anamnestic information, D. repens was suspected, but as the sample represented an immature nematode lacking diagnostic morphological features, the specimen was submitted for molecular identification.
Molecular identification
For a specific diagnosis, DNA was isolated from a blood sample containing mf from the human patient and from the ethanol-preserved worm specimen from the second canine case. DNA of three adult D. ursi specimens, from three different brown bears from eastern Finland, was used as a reference. Partial mitochondrial sequences of the cytochrome c oxidase subunit 1 (cox1) gene and 12S ribosomal DNA (rDNA) were amplified using previously reported filarioid-specific primers [37, 38]. Standard Sanger sequencing of the amplicons confirmed the causative agent to be D. repens in both the human and canine cases. The sequences (cox1, 649 bp; 12S rDNA, 468 bp) of the human patient were identical with several published sequences of D. repens in GenBank (e.g. cox1, Estonian canine isolate, accession no. KR780980; 12S rDNA, Russian human isolate, KM205372). The cox1 sequence of the canine specimen and that of the human patient were identical, and the identity in 12S rDNA was 99%. In both sequence regions of the human and canine specimens, the identity to D. ursi was 92%. The sequence data are available in the GenBank database under the accession numbers KY828978–KY828986.
